Detoxication And Mechanism Of Chicken Egg Yolk Immunoglobulins On Aflatoxin B₁-induced Toxicity

Aflatoxins are secondary metabolites produced by Aspergillus flavus or Aspergillus parasiticus,and widely distributed in various food products and feed.Among the aflatoxins,aflatoxin B₁（AFB₁）is the most hepatotoxic,genotoxic and immunotoxic to human and animal.Various neutralizing agents and mechanism of AFB₁ toxicity have been studied.Egg yolk immunoglobulins（IgY）,a kind of antibody that is found in high concentrations in chicken egg yolk,has been recognized as a lower cost and convenient source of polyclonal antibody.IgY is composed of two L and two H chains with two antigen binding sites like mammalian counterpart IgG.IgY has been used in the treatment of various gastroenteric infectious diseases,and against macromolecular protein toxins.However,the detoxicattion effect and mechanism of IgY on small molecular toxins have not yet been reported.In this study,detoxication and mechanism of IgY on AFB₁-induced toxicity was firstly investigated.Chickens were immunized with the artificial hapten of AFB₁-GA-BSA prepared to abtain highly purified and specificity anti-AFB₁ IgY.To evaluate the protection of IgY against AFB₁-induced cytotoxicity,dysfunction and genotoxicity,the detoxification of IgY was assessed against AFB₁ using human L-02 hepatocytes as model.The protection of IgY also was assessed against AFB₁-induced toxicity using human hepatic cancer cell HepG2,colon cancer cell HTC-8,and trophoblasts Swan 71.Pathophysiological and metabolites experiments was designed to evaluate protection and mechanism of IgY against AFB₁-induced genotoxicity and oxidative damage in animal level.The main studies and results are as follows:1.Production of highly purified and specificity anti-AFB₁ IgYTo prepare the artificial antigen,a novel derivatization method was used to modify the hapten of AFB₁,by coupling it with a carrier protein via the active ester synthesis method.Chickens were immunized with AFB₁-GA-BSA to abtain anti-AFB₁ IgY.The oximation reaction was used to produce the artificial antigen of zearalenone.And anti-ZEN IgY were obtained by immunizing with ZEN-COM-BSA which was prepared by the active ester method.The IgY was purified by a combination of several purification technology including water-soluble fraction under acidic conditions,filtration and ammonium sulfate precipitation method.The IgY purity was identified by SDS-PAGE and quantified with ImageJ software.The purity of IgY was estimated to be⁹4%.The generated time of IgY are different due to different antigens,and the generated time of anti-AFB₁ IgY was shorter than anti-ZEN IgY after primary immunization.Anti-AFB₁ IgY was specific to AFB₁ detected by indirect competitive enzyme-linked immunosorbent assay（ic-ELISA）was established with LOD（0.06 ng/mL）,a sensitive half-maximal inhibitory concentration(IC_50,2.4 ng/mL)and dynamic working range（0.13–43.0 ng/mL）.Anti-ZEN IgY was analysed in same way with LOD（9.27 ng/mL）,half-maximal inhibitory concentration(IC_50,83.76 ng/mL)and dynamic working range（13.78-508.85 ng/mL）.They all were sensitive enough to recognize and bind antigen.2.IgY reduces AFB₁-induced cytotoxicity,cellular dysfunction,and genotoxicity in human L-02 hepatocytesThe anti-AFB₁ IgY obtained reduced AFB₁-induced cytotoxicity,cellular dysfunction,and genotoxicity by protecting cells against apoptotic body formation and DNA strand breaks,preventing G2/M phase cell cycle arrest,reducing AFB₁-DNA adduct and ROS production,and the mitochondrial membrane potential in a strong dose-dependent manner.Anti-AFB₁IgY inhibited the AFB₁-induced expression of proteins related to anti-apoptotic,pro-apoptotic,and antioxidative processes in a dose-dependent manner,but anti-AFB₁ IgY sources may not directly affect the level of these protein expression.We also found AFB₁-DNA adduct formation were gradually decreased in L-02 cells with increasing dosage of anti-AFB₁ IgY.The results indicating that AFB₁ could not entry cell after combine with anti-AFB₁IgY.This is the first time that IgY sources have detoxification properties in mycotoxin（small molecular toxins）-induced toxicity in vitro.3.IgY reduces AFB₁-induced toxicity in other cell modelThe principal target organ of AFB₁ is liver,gut,and reproductive system.Human hepatic cancer cell HepG2,colon cancer cell HTC-8,and trophoblasts Swan 71 as cell model was used to investigate whether anti-AFB₁ IgY could inhibit AFB₁-induced toxicity.The effect of anti-AFB₁ IgY against AFB₁-induced cell survival,apoptosis,migration and invasion was investigated.Our experimental results showed that anti-AFB₁ IgY reduces AFB₁-induced cell apoptosis and maintaining cell migration and invasion in a strong dose-dependent manner.These experiments demonstrated that the anti-AFB₁ IgY also has broad protection effects to reduces AFB₁-induced toxicity in other cell model.We speculate that anti-AFB₁ IgY could protects liver,gut,and reproductive system from aflatoxin B₁-induced toxicity damage via oral administration of specific anti-AFB₁ IgY with AFB₁.4.IgY prevents rat liver from aflatoxin B₁-induced genotoxicity and oxidative damageDifferent concentrations of anti-AFB₁ IgY and AFB₁（2.5μM）were mixed in vitro to evaluate binding activity of anti-AFB₁ IgY to AFB₁ by monitoring chang of AFB₁fluorescence,and a model assuming a 1:80 ratio of anti-AFB₁ IgY to AFB₁ binding.The protection of anti-AFB₁ IgY against AFB₁-induced genotoxicity and oxidative damage in the rat liver model were investigated in pathophysiological and metabolites study.Our results revealed that AFB₁ induced significant oxidative damage markers,as well as AFB₁-induced protein expression in antioxidant,pro-and anti-apoptosis processes in rat liver.These effects could be significantly inhibited by co-gavage with anti-AFB₁ IgY in a dose-dependent manner.However,anti-AFB₁ IgY did not significantly induce hepatic CAT and SOD1.The effects were further verified in metabolite study,in which the influence of anti-AFB₁ IgY on the absorption and toxicity of AFB₁ in rats was investigated.Middle and high dose of anti-AFB₁ IgY reduced hepatic DNA adduction by 43.3%and 52.9%,AFB₁-albumin adducts by10.5%and 21.1%,and the major AFB₁-N⁷-guanine urinary adduct by 19.6%and 34.4%,respectively.These metabolites of AFB₁ are well known markers of AFB₁-induced genotoxicity.The faeces of high dose anti-AFB₁ IgY co-gavaged rats contained approximately 2-fold higher AFB₁ equivalents at 6 h after ingestion than AFB₁ group faeces,indicating IgY inhibited AFB₁ uptake.These results are consistent with a mechanism involving complex-mediated reduction of AFB₁ uptake,and IgY prevents rat liver from aflatoxin B₁-induced genotoxicity and oxidative damage.In conclusion,highly purified and specificity anti-AFB₁ IgY was successfully obtained from chickens in this study.The anti-AFB₁ IgY bound AFB₁ could not enter cells to reduces AFB₁-induced cytotoxicity,cellular dysfunction,and genotoxicity in vitro.Formation of a non-covalent complex between AFB₁ and anti-AFB₁ IgY could reduce the absorption of AFB₁ to prevents rat liver from aflatoxin B₁-induced genotoxicity and oxidative damage.This is the first investigation into the toxicity-reducing effect of specific IgY on small molecule toxin damage,which will be beneficial for the development of antibodies as detoxication agents.